Volume 19, Issue 5, Pages 652-663 (May 2011) c-Raf, but Not B-Raf, Is Essential for Development of K-Ras Oncogene-Driven Non- Small Cell Lung Carcinoma  Rafael B. Blasco, Sarah Francoz, David Santamaría, Marta Cañamero, Pierre Dubus, Jean Charron, Manuela Baccarini, Mariano Barbacid  Cancer Cell  Volume 19, Issue 5, Pages 652-663 (May 2011) DOI: 10.1016/j.ccr.2011.04.002 Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 1 K-RasG12V Induces NSCLCs in the Absence of Erk1 or Erk2 (A) The left panel shows survival of K-Ras+/G12V;Erk1+/+ (n = 17) (open circles) and K-Ras+/G12V;Erk1−/− (n = 14) (solid circles) mice treated with Ad-Cre at 8 weeks of age. The right panel illustrates survival of K-Ras+/G12V;Erk2+/+ (n = 19) (open circles) and K-Ras+/G12V;Erk2lox/lox (n = 32) (solid circles) mice treated with Ad-Cre at 8 weeks of age. (B) The left panel shows the number of tumors, classified by grade (I–IV), observed in K-Ras+/G12V;Erk1+/+ (n = 5) (open bars) and K-Ras+/G12V;Erk1−/− (n = 5) (solid bars) mice sacrificed 6 months after Ad-Cre treatment. The right panel illustrates the number of tumors, classified by grade (I–IV), observed in K-Ras+/G12V;Erk2+/+ (n = 5) (open bars) and K-Ras+/G12V;Erk2lox/lox (n = 5) (solid bars) mice sacrificed 6 months after Ad-Cre treatment. Error bars indicate mean ± SD. p values were calculated according to Student's t test. (C) Western blot analysis of pErk1/2, Erk1/2, pRsk, and Rsk expression in lysates prepared from individual tumors of the indicated genotype collected 8 months after Ad-Cre treatment. Gapdh is shown as a loading control. Migration of the above proteins is indicated by arrowheads. See also Figure S1. Cancer Cell 2011 19, 652-663DOI: (10.1016/j.ccr.2011.04.002) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 2 Complete Ablation of Erk1/2 Kinases Prevents Induction of NSCLCs by a Resident K-RasG12V Oncogene (A) Survival of K-Ras+/G12V;Erk1+/+;Erk2+/+ (n = 14) (open circles) and K-Ras+/G12V;Erk1−/−;Erk2lox/lox (n = 11) (solid circles) mice treated with Ad-Cre at 8 weeks of age. (B) Whole-mount X-Gal staining of lung sections collected from mice with the indicated genotypes 6 months after Ad-Cre treatment. β-Geo positive cells identified by X-Gal staining (blue color) correspond to cells expressing K-RasG12V. Scale bars, 5 mm. (C) Number of tumors, classified by grade (I–IV), observed in K-Ras+/G12V;Erk1+/+;Erk2+/+ (n = 7) (open bars) and K-Ras+/G12V;Erk1−/−;Erk2lox/lox (n = 10) (solid bars) mice sacrificed 6 months after Ad-Cre treatment. Error bars indicate mean ± SD. p values were calculated according to Student's t test. (D) The top panel shows Southern blot analysis of genomic DNA isolated from individual tumors of K-Ras+/G12V;Erk1−/−;Erk2lox/lox mice 8 months after Ad-Cre treatment. DNAs were digested with KpnI and probed with a 450 bp DNA fragment derived from the third intron just outside the floxed sequences. Migration of the unrecombined Erk2lox allele (4.3 kbp) and the ablated Erk2− allele (2.4 kbp) is indicated by arrowheads. The bottom panel shows western blot analysis of Erk1 and Erk2 expression in lysates obtained from individual tumors collected 8 months after Ad-Cre treatment of K-Ras+/G12V;Erk1+/+;Erk2+/+ and K-Ras+/G12V;Erk1−/−;Erk2lox/lox mice. The presence of Erk2 in tumors of Ad-Cre treated K-Ras+/G12V;Erk1−/−;Erk2lox/lox mice, due to incomplete cleavage of the Erk2lox allele, indicates that Erk2 is essential for tumor development. Gapdh was used as loading control. Migration of the above proteins is indicated by arrowheads. (E) Survival of Erk1+/+;Erk2+/+;RERTert/ert (open circles) and Erk1−/−;Erk2lox/lox;RERTert/ert (solid circles) mice fed ad libitum a tamoxifen-containing diet to activate the knocked in CreERT2 recombinase encoded by the RERTert alleles. See also Figure S2. Cancer Cell 2011 19, 652-663DOI: (10.1016/j.ccr.2011.04.002) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 3 K-RasG12V Induces NSCLCs in the Absence of Mek1 or Mek2 (A) The left panel shows survival of K-Ras+/G12V;Mek1+/+ (n = 20) (open circles) and K-Ras+/G12V;Mek1lox/lox (n = 14) (solid circles) mice treated with Ad-Cre at 8 weeks of age. The right panel illustrates survival of K-Ras+/G12V;Mek2+/+ (n = 20) (open circles) and K-Ras+/G12V;Mek2−/− (n = 19) (solid circles) mice treated with Ad-Cre at 8 weeks of age. (B) The left panel shows the number of tumors, classified by grade (I–IV), observed in K-Ras+/G12V;Mek1+/+ (n = 7) (open bars) and K-Ras+/G12V;Mek1lox/lox (n = 6) (solid bars) mice sacrificed 6 months after Ad-Cre treatment. The right panel illustrates the number of tumors, classified by grade (I–IV), observed in K-Ras+/G12V;Mek2+/+ (n = 5) (open bars) and K-Ras+/G12V;Mek2−/− (n = 5) (solid bars) mice sacrificed 6 months after Ad-Cre treatment. Error bars indicate mean ± SD. p values were calculated according to Student's t test. (C) Western blot analysis of pMek, Mek1, Mek2, pErk1/2, and Erk1/2 expression in lysates derived from individual tumors of the indicated genotype collected 8 months after Ad-Cre treatment. Gapdh was used as loading control. Migration of the above proteins is indicated by arrowheads. See also Figure S3. Cancer Cell 2011 19, 652-663DOI: (10.1016/j.ccr.2011.04.002) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 4 Elimination of Mek1/2 Kinases Prevents Induction of NSCLCs by an Endogenous K-RasG12V Oncogene (A) Survival of K-Ras+/G12V;Mek1+/+;Mek2+/+ (n = 10) (open circles) and K-Ras+/G12V;Mek1lox/lox;Mek2−/− (n = 14) (solid circles) mice treated with Ad-Cre at 8 weeks of age. (B) Whole-mount X-Gal staining of lung sections collected from mice with the indicated genotypes 6 months after Ad-Cre treatment. β-Geo positive cells identified by X-Gal staining (blue color) correspond to cells expressing K-RasG12V. Scale bars, 5 mm. (C) Number of tumors, classified by grade (I–IV), observed in K-Ras+/G12V;Mek1+/+;Mek2+/+ (n = 6) (open bars) and K-Ras+/G12V;Mek1lox/lox;Mek2−/− (n = 8) (solid bars) mice. Error bars indicate mean ± SD. p values were calculated according to Student's t test. (D) The top panel shows Southern blot analysis of genomic DNA isolated from individual tumors of K-Ras+/G12V;Mek1lox/lox;Mek2−/− mice 8 months after Ad-Cre treatment. DNAs were digested with HindIII and probed with a 680 bp DNA fragment obtained from a region downstream from the second loxP site. The migration of the unrecombined Mek1lox allele (1.7 kbp) and the Mek1– allele (1.5 kbp) is indicated by arrowheads. The bottom panel shows western blot analysis of Mek1 and Mek2 expression in lysates obtained from individual tumors collected 8 months after Ad-Cre treatment of K-Ras+/G12V;Mek1+/+;Mek2+/+ and K-Ras+/G12V;Mek1lox/lox;Mek2−/− mice. The presence of Mek1 in tumors of Ad-Cre treated K-Ras+/G12V;Mek1lox/lox;Mek2−/− mice, due to partial cleavage of the Mek1lox allele, indicates that Mek1 is essential for tumor development. Gapdh was used as loading control. Migration of the above proteins is indicated by arrowheads. (E) Survival of Mek1+/+;Mek2+/+;RERTert/ert (n = 6) (open circles) and Mek1lox/lox;Mek2−/−;RERTert/ert (n = 6) (solid circles) mice fed ad libitum a tamoxifen-containing diet to activate the knocked in CreERT2 recombinase encoded by the RERTert alleles. See also Figure S4. Cancer Cell 2011 19, 652-663DOI: (10.1016/j.ccr.2011.04.002) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 5 B-Raf Is Not Required for K-Ras+/G12V-Induced NSCLCs in Mice (A) Survival of K-Ras+/G12V;B-Raf+/+ (n = 28) (open circles) and K-Ras+/G12V;B-Raflox/lox (n = 25) (solid circles) mice treated with Ad-Cre at 8 weeks of age. (B) Whole-mount X-Gal staining of lung sections collected from mice with the indicated genotypes 6 months after Ad-Cre treatment. β-Geo positive cells identified by X-Gal staining (blue color) correspond to cells expressing K-RasG12V. Scale bars, 5 mm. (C) Number of tumors, classified by grade (I–IV), observed in K-Ras+/G12V;B-Raf+/+ (n = 5) (open bars) and K-Ras+/G12V;B-Raflox/lox (n = 5) (solid bars) mice. Error bars indicate mean ± SD. p values were calculated according to Student's t test. (D) Western blot analysis of B-Raf, c-Raf, A-Raf, pMek, Mek1, pErk1/2, and Erk1/2 expression in lysates derived from individual tumors of the indicated genotype collected 8 months after Ad-Cre treatment. Gapdh was used as loading control. Migration of the above proteins is indicated by arrowheads. (E) Tumors retained phosphorylated Erk expression in the absence of B-Raf. Hematoxylin and eosin (H&E) (left panels) and immunohistochemical staining of consecutive paraffin-fixed sections using anti-B-Raf (center panels) and anti-pErk (right panels) antibodies. Sections were obtained from lungs of Ad-Cre treated K-Ras+/G12V;B-Raf+/+ (top panels) and K-Ras+/G12V;B-Raflox/lox (bottom panels) mice. Insets show detail of larger areas. Scale bars, 0.5 mm (main field) and 0.02 mm (inset). See also Figure S5. Cancer Cell 2011 19, 652-663DOI: (10.1016/j.ccr.2011.04.002) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 6 c-Raf Is Essential for K-Ras+/G12V-Induced NSCLCs in Mice (A) Survival of K-Ras+/G12V;c-Raf+/+ (n = 22) (open circles) and K-Ras+/G12V;c-Raflox/lox (n = 23) (solid circles) mice treated with Ad-Cre at 8 weeks of age. (B) Whole-mount X-Gal staining of lung sections collected from mice with the indicated genotypes 6 months after Ad-Cre treatment. β-Geo positive cells identified by X-Gal staining (blue color) correspond to cells expressing K-RasG12V. Scale bars, 5 mm. (C) Number of tumors, classified by grade (I–IV), observed in K-Ras+/G12V;c-Raf+/+ (n = 8) (open bars) and K-Ras+/G12V;c-Raflox/lox (n = 8) (solid bars) mice. Error bars indicate mean ± SD. p values were calculated according to Student's t test. (D) The top panel shows Southern blot analysis of DNA isolated from individual tumors obtained from K-Ras+/G12V;c-Raflox/lox mice infected with Ad-Cre particles at 8 weeks of age. Tumor DNAs were digested with PstI. The sizes of the diagnostic DNA fragments for c-Raflox and c-Raf− alleles are indicated. The bottom panel illustrates western blot analysis of c-Raf expression in lysates obtained from individual tumors collected 8 months after Ad-Cre treatment of K-Ras+/G12V;c-Raf+/+ and K-Ras+/G12V;c-Raflox/lox mice. The presence of c-Raf in tumors of Ad-Cre treated K-Ras+/G12V;c-Raflox/lox mice is due to incomplete cleavage of the c-Raflox allele. These results indicate that c-Raf is essential for tumor development. Gapdh was used as loading control. Migration of the above proteins is indicated by arrowheads. See also Figure S6. Cancer Cell 2011 19, 652-663DOI: (10.1016/j.ccr.2011.04.002) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 7 Adult Mice Tolerate Widespread Ablation of B-Raf and c-Raf Alleles (A) Southern blot analysis of DNA isolated from tissues of c-Raflox/lox;RERTert/ert mice fed ad libitum with a tamoxifen diet for 3 months (P30–P120). DNA was digested with PstI and probed with a 854 bp DNA fragment corresponding to sequences located in intron 4 downstream from the second loxP site. Migration of the ablated c-Raf− allele and the unrecombined c-Raflox allele is indicated by arrowheads. The sizes of the diagnostic DNA fragments for these alleles are also indicated. Whereas some tissues such as testis (Te), ovaries (Ov), heart (Ht), and muscle (Mu) display partial c-Raflox cleavage (from 50% to 70%), the majority of the tissues, including lung (Lu), stomach (St), colon (Co), skin (Sk), intestine (In), liver (Li), kidney (Ki), pancreas (Pa), and spleen (Sp), showed complete or almost complete excision. Brain tissue (Br) served as negative control because tamoxifen crosses the brain-blood barrier with limited efficiency. (B) Southern blot analysis of DNA isolated from tissues of B-Raflox/lox;c-Raflox/lox;RERTert/ert mice fed ad libitum with a tamoxifen diet for 3 months (P30–P120). DNA samples were digested with HindIII and probed with a 422 bp DNA fragment corresponding to sequences located in intron 12 upstream from the first loxP site. Migration of the ablated B-Raf null (B-Raf−) and c-Raf null (c-Raf−) alleles as well as of the unrecombined B-Raflox and c-Raflox alleles is indicated by arrowheads. The sizes of the diagnostic DNA fragments for these alleles are also indicated. The top panel shows B-Raflox alleles that are almost completely recombined in lung (Lu), stomach (St), colon (Co), kidney (Ki), pancreas (Pa), spleen (Sp), and thymus (Th) and only partially cleaved in testis (Te), ovaries (Ov), and heart (Ht). The bottom panel illustrates c-Raflox alleles that were partially excised in testis (Te), ovaries (Ov), heart (Ht), lung (Lu), stomach (St), and colon (Co) and completely recombined in kidney (Ki), pancreas (Pa), spleen (Sp), and thymus (Th). Brain tissue (Br) served as negative control. See also Figure S7. Cancer Cell 2011 19, 652-663DOI: (10.1016/j.ccr.2011.04.002) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 8 B-Raf and c-Raf Are Not Essential for Proliferation and Immortalization of Primary MEFs Driven by Wild-Type or Oncogenic K-Ras Signaling (A) Western blot analysis of B-Raf and c-Raf protein expression in primary MEFs derived from (top panel) three independent K-Ras+/G12V;RERTert/ert, K-Ras+/G12V;B-Raflox/lox;RERTert/ert, K-Ras+/G12V;c-Raflox/lox;RERTert/ert, and K-Ras+/G12V;B-Raflox/lox;c-Raflox/lox;RERTert/ert embryos, and (bottom panel) three independent K-Ras+/+;RERTert/ert, K-Ras+/+;B-Raflox/lox;RERTert/ert, K-Ras+/+;c-Raflox/lox;RERTert/ert, and K-Ras+/+;B-Raflox/lox;c-Raflox/lox;RERTert/ert embryos. These MEFs were incubated in DMEM supplemented with 10% FBS in the presence of 4OHT for 5 days to activate expression of the resident K-Ras+/G12V oncogene and to eliminate the c-Raflox (c-RafΔ) and/or B-Raflox (B-RafΔ) conditional alleles. Gapdh served as loading control. Migration of the corresponding proteins is indicated by arrowheads. (B) Growth curves of the primary MEFs (n = 3) described above. MEFs were grown in DMEM supplemented with 10% FBS and 600 nM 4OHT for 5 days before seeding. Results are shown in arbitrary absorbance units. Left panel shows K-Ras+/G12V;RERTert/ert (solid circles), K-Ras+/G12V;B-RafΔ/Δ;RERTert/ert (open squares), K-Ras+/G12V;c-RafΔ/Δ;RERTert/ert (open circles), and K-Ras+/G12V;B-RafΔ/Δ;c-RafΔ/Δ;RERTert/ert (open triangles). Right panel illustrates K-Ras+/+;RERTert/ert (solid circles), K-Ras+/+;B-RafΔ/Δ;RERTert/ert (open squares), K-Ras+/+;c-RafΔ/Δ;RERTert/ert (open circles), and K-Ras+/+;B-RafΔ/Δ;c-RafΔ/Δ;RERTert/ert (open triangles). Error bars indicate mean ± SD. (C) The left panel shows western blot analysis of B-Raf and c-Raf protein expression in primary MEFs derived from five independent K-Ras+/G12V;B-Raflox/lox;RERTert/ert, K-Ras+/G12V;c-Raflox/lox;RERTert/ert, and K-Ras+/G12V;B-Raflox/lox;c-Raflox/lox;RERTert/ert embryos. Samples from two K-Ras+/G12V;RERTert/ert embryos are also shown. MEFs were incubated in DMEM supplemented with 2% FBS in the presence of 4OHT for 5 days to activate expression of the resident K-RasG12V oncogene and to eliminate the c-Raflox (c-RafΔ) and/or B-Raflox (B-RafΔ) conditional alleles. Gapdh served as loading control. Migration of the corresponding proteins is indicated by arrowheads. The right panel shows growth curves of the primary MEFs (n = 5) described above. MEFs were grown in DMEM supplemented with 2% FBS and 600 nM 4OHT. K-Ras+/G12V;RERTert/ert (solid circles), K-Ras+/G12V;B-RafΔ/Δ;RERTert/ert (open squares), K-Ras+/G12V;c-RafΔ/Δ;RERTert/ert (open circles), and K-Ras+/G12V;B-RafΔ/Δ;c-RafΔ/Δ;RERTert/ert (open triangles). Error bars indicate mean ± SD. (D) Immortalization of the primary MEFs described in (B) following a 3T3 protocol (n = 3). Cells were cultivated in DMEM supplemented with 10% FBS and 600 nM 4OHT. Symbols are those described in (B). Error bars indicate mean ± SD. See also Figure S8. Cancer Cell 2011 19, 652-663DOI: (10.1016/j.ccr.2011.04.002) Copyright © 2011 Elsevier Inc. Terms and Conditions